DOCSLIB.ORG

Characterization of Yoaa As It Relates to DNA Replication and Repair

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Characterization of Yoaa As It Relates to DNA Replication and Repair Characterization of YoaA as it Relates to DNA Replication and Repair Senior Thesis Presented to The Faculty of the School of Arts and Sciences Brandeis University Undergraduate Program in Biology Susan T. Lovett, Advisor In partial fulfillment of the requirements of the degree of Bachelor of Science by Gabriela Giordano May 2021 Copyright by Gabriela Giordano Acknowledgments I would like to thank my advisor Dr. Susan T. Lovett for all of the direction she provided throughout this project. I greatly appreciate the trust she put in me as a researcher in her lab and admire the supportive and engaging lab environment she has created. A big thank you goes to my mentor Vincent A. Sutera, whom I owe for almost everything I’ve learned as a member of the Lovett Lab. I appreciate him taking me on as a mentee and continuing to have faith in me until the end. He has always given me the opportunity to learn something new when I needed a challenge and has pushed me to become more independent in my work. A special thanks goes to David Glass who has answered every question I’ve ever had, taught me how to use the multiplate reader, and was always there for a good laugh. A final thank you goes to Yonatan Zur, without whom I could have never made it this far. Whether it was showing me where items in the lab are kept, taking plates out of the incubator, Sunday Zoom calls about experiments, or Saturday thesis work outside, I have always been able to count on Yonatan and could not possibly thank him enough. ii Table of Contents Acknowledgments……………………………………………………………………..………….ii Abstract……………………..………………………………………………………….…………iii Introduction………………………………………………………………………………………..1 Results………………………………………………………………………………………..…..10 Discussion……………………………………………………………………………………..…14 Materials and Methods…………………………………………………………………………...17 Bibliography……………………………………………………………………………….…….29 i Abstract Effective DNA replication and repair is crucial to the survival of all organisms and necessitates the cooperation of a variety of macromolecules. In Escherichia coli, the DNA Polymerase III holoenzyme is one of these macromolecules which, along with being the main contributor to DNA replication, is known to participate in other interactions which aid in the repair of damaged DNA. One such interaction is with YoaA, a putative helicase found to promote tolerance to replication inhibiting factors. The interaction between YoaA and the DNA Polymerase III holoenzyme takes place at HolC, one of the holoenzyme’s protein subunits. This project seeks to further characterize the interaction between YoaA and HolC while also attempting to identify the types of DNA damage and repair to which YoaA responds. Through Yeast Two-Hybrid, I have shown that the C-terminus of YoaA is the region responsible for HolC binding. Additionally, E. coli growth assays show that mutations in the C-terminus of YoaA may alter the protein’s function in vivo. A variety of phenotypic assays have suggested that YoaA does not exhibit a significant response to recombination events or point mutations. ii Introduction to DNA Repair and YoaA DNA replication is one of the most fundamental biological processes present in all living organisms. Given that cells contain such large quantities of genetic material, it is inevitable that DNA will occasionally contain mistakes. Whether these mistakes are a result of errors in replication or of external mutagens, they pose a problem to further DNA replication. Within cells exist mechanisms capable of identifying such mistakes, recruit the proper repair units, and correct the mistakes to continue replication of DNA. As the correct replication and repair of DNA is vital to the survival of all organisms, including humans, it is the focus of a great deal of scientific research. Escherichia coli (E. coli) has served as a model organism to study the processes involved in replication and repair because it utilizes several mechanisms and proteins analogous to those of humans. Though much has been learned about DNA replication and repair, there is still a significant amount that is unknown about the intricacies of these biological processes, and research continues to work toward better understanding the cellular participants and mechanisms involved. One of the relatively unexplored areas of this field deals with the moment at which the cellular replication machinery identifies a mistake in a DNA sequence and recruits those proteins whose function is to repair the mistake. Several proteins are required to facilitate DNA repair, some of which are helicases, which unwind DNA structures in a multitude of situations that require unencumbered DNA for replication. The purpose of my research is to further understand what happens when DNA replication encounters a mistake, with a specific focus on YoaA, a putative helicase that is thought to participate in this process. Little is known about YoaA’s role in DNA repair or about what circumstances result in its recruitment by the cell. My research seeks to further characterize YoaA’s behavior and function in the replication and repair of E. coli DNA. 1 DNA Polymerase III In E. coli, the DNA polymerase III holoenzyme (Pol III) is responsible for catalyzing the majority of DNA replication. Pol III is encoded by nine different genes that give rise to various subunits (see figure 1). The core complex of Pol III, which exhibits both polymerase and exonuclease activity, is comprised of an α, ε, and θ subunit (encoded by dnaE, dnaQ, and holE respectively). The core complex associates with the clamp loader complex that is responsible for loading the β clamp onto the DNA, the interactions of which work to stabilize the polymerases binding to DNA during replication. Accessory to the Pol III τ τ clamp loader (encoded by holA, holB, and dnaX) are two additional protein subunits: χ (encoded by holC) and ψ δ’ (encoded by holD) (Duigou 1). Though much remains δ unknown about the functions of the HolC and HolD Figure 1: DNA Polymerase III Holoenzyme subunits, there is new evidence to show that both participate (Created with BioRender.com) in coordinating DNA repair when Pol III runs into damaged DNA during replication (Brown et al 2015). HolC and HolD While the clamps and clamp loaders involved in DNA replication are universally conserved, with homologs across a variety of eukaryotes, neither HolC nor HolD has been found to have homologous eukaryotic counterparts and are observed only in proteobacteria. This lack of conservation aligns with the non-essential nature of HolC in E. coli and contributes to novel understandings of DNA replication. HolC participates in a variety of processes that assist Pol III 2 activity. One of this protein’s primary interactions is with single-strand DNA binding protein (SSB) and through its binding to SSB, it aids the polymerization of substrates coated with SSB. The HolC-HolD complex increases the clamp-loader’s affinity to primer-template DNA and is thought to stabilize the interaction between Pol III for this reason (Duigou 2). This effect of the HolC-HolD complex functions to promote clamp-loader activity and increase the overall processivity of Pol III. Additionally, HolC assists in displacing primase from RNA primers. It accomplishes this by binding to SSB, which is initially bound to primase and is then transferred to HolC at the primer-template junction (Duigou 2014). Because of its strong connection to SSB, it is believed that HolC acts primarily on the SSB-coated lagging-strand DNA template. This conclusion supports HolC’s coordination of DNA repair as the lagging strand is often subject to the formation and accumulation of DNA duplexes and is frequently in need of DNA repair. Codependence of HolC and YoaA In addition to assisting in DNA replication, HolC has been shown (Brown et al. 2015) to interact with YoaA, a recently discovered putative helicase thought to be involved in DNA repair. The dependence of YoaA and HolC on each other was initially observed using Azidothymidine (AZT) sensitivity assays which tested E. coli’s survival when either holC or yoaA had been deleted. AZT is a chain-terminating nucleoside analog that causes the accumulation of single-stranded DNA gaps during replication. By challenging E. coli with the deletion of holC and yoaA, the dependence of the proteins on each other could be more closely examined. It was found that, in both strains, bacteria exhibited significant sensitivity to AZT. Additionally, AZT sensitivity was not suppressed by overexpressing the other gene in a plasmid vector, suggesting that YoaA and HolC are codependent and that their interaction is necessary for normal cell growth (Brown, et al 2015). 3 Physical Interaction Between YoaA and HolC Having confirmed the codependence of YoaA and HolC, research has shifted toward examining the physical interaction between the two proteins. A common way to screen for protein binding is through Yeast Two-Hybrid analysis, which is based in the GAL4/UAS system. In this system, one protein of interest is fused to the binding domain (bait) and the other to the activation domain (prey). The binding domain binds the UAS promoter sequence, and, when there is bait-prey interaction, reconstitutes a transcription factor that recruits RNA Polymerase II and allows for normal transcription (Brückner 2009). Therefore, if the two proteins of interest interact with one another, there will be observable growth. In Yeast-Two Hybrid analysis, the relative strength of protein binding can be determined using different growth media: growth on histidine dropout media indicates weak interaction while growth on adenine dropout indicates strong interaction. This type of analysis was performed with YoaA and HolC (Brown, et al. 2015) and, upon observing growth on histidine dropout media, it was determined that the two proteins physically interact. Specifics of YoaA-HolC Binding Site Upon confirming that HolC and YoaA proteins interact and play a role in DNA repair, the next step has been to narrow in on the specificities of the binding between HolC and YoaA.
Load more

Recommended publications
  • Chi Subunit of Polymerase III Holoenzyme May Have Function in Addition to Facilitating DNA Replication
    Chi Subunit of Polymerase III Holoenzyme May Have Function in Addition to Facilitating DNA Replication Master’s Thesis Presented to The Faculty of the Graduate School of Arts and Sciences Brandeis University Department of Biochemistry Dr. Susan Lovett, Advisor In Partial Fulfillment of the Requirements for the Degree Master of Science in Biochemistry by Taku Harada May 2018 Copyright by Taku Harada © 2018 Acknowledgments I would like to thank my advisor Dr. Susan Lovett. Thank you for sharing this opportunity to explore the E. coli genome with you. I had a wonderful experience. Along the way, your unwavering support and encouragement was irreplaceable. I am greatly fortunate and appreciative. Thank you to my mentor Dr. Alex Ferazzoli. No amount of words could ever describe the gratitude I have for you. You were a mentor for me in science and life. Thank you for always supporting and encouraging me to learn even if, at times, it meant failure. I attribute my success to your investment and confidence in me. Most importantly, your enthusiasm and joyous personality is inspirational and made every day a good day. Thank you to Ariana, Dr. Cooper, Laura, Vinny, Julie, McKay and everyone who worked in the Lovett lab during my stay. You all welcomed me in and provided a supportive environment that extended beyond the lab walls. I am very fortunate to have worked with all of you. Thank you to all my friends. Special thanks Adib, Eli, Jessie, and Rich. My four years at Brandeis have been phenomenal because of your support and motivation. I look forward to many more years of friendship.
    [Show full text]
  • DNA POLYMERASE III HOLOENZYME: Structure and Function of a Chromosomal Replicating Machine
    Annu. Rev. Biochem. 1995.64:171-200 Copyright Ii) 1995 byAnnual Reviews Inc. All rights reserved DNA POLYMERASE III HOLOENZYME: Structure and Function of a Chromosomal Replicating Machine Zvi Kelman and Mike O'Donnell} Microbiology Department and Hearst Research Foundation. Cornell University Medical College. 1300York Avenue. New York. NY }0021 KEY WORDS: DNA replication. multis ubuni t complexes. protein-DNA interaction. DNA-de penden t ATPase . DNA sliding clamps CONTENTS INTRODUCTION........................................................ 172 THE HOLO EN ZYM E PARTICL E. .......................................... 173 THE CORE POLYMERASE ............................................... 175 THE � DNA SLIDING CLAM P............... ... ......... .................. 176 THE yC OMPLEX MATCHMAKER......................................... 179 Role of ATP . .... .............. ...... ......... ..... ............ ... 179 Interaction of y Complex with SSB Protein .................. ............... 181 Meclwnism of the yComplex Clamp Loader ................................ 181 Access provided by Rockefeller University on 08/07/15. For personal use only. THE 't SUBUNIT . .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 182 Annu. Rev. Biochem. 1995.64:171-200. Downloaded from www.annualreviews.org AS YMMETRIC STRUC TURE OF HOLO EN ZYM E . 182 DNA PO LYM ER AS E III HOLO ENZ YME AS A REPLIC ATING MACHINE ....... 186 Exclwnge of � from yComplex to Core .................................... 186 Cycling of Holoenzyme on the LaggingStrand
    [Show full text]
  • Connecting Replication and Repair: Yoaa, a Helicase-Related Protein, Promotes Azidothymidine Tolerance Through Association with Chi, an Accessory Clamp Loader Protein
    RESEARCH ARTICLE Connecting Replication and Repair: YoaA, a Helicase-Related Protein, Promotes Azidothymidine Tolerance through Association with Chi, an Accessory Clamp Loader Protein Laura T. Brown, Vincent A. Sutera, Jr., Shen Zhou, Christopher S. Weitzel¤, Yisha Cheng, Susan T. Lovett* a11111 Department of Biology and Rosenstiel Basic Medical Sciences Research Center MS029, Brandeis University, Waltham, Massachusetts, United States of America ¤ Current address: Department of Biochemistry, School of Molecular and Cellular Biology, University of Illinois Urbana-Champaign, Urbana, Illinois, United States of America * [email protected] OPEN ACCESS Abstract Citation: Brown LT, Sutera VA, Jr., Zhou S, Weitzel CS, Cheng Y, Lovett ST (2015) Connecting Elongating DNA polymerases frequently encounter lesions or structures that impede prog- Replication and Repair: YoaA, a Helicase-Related ress and require repair before DNA replication can be completed. Therefore, directing repair Protein, Promotes Azidothymidine Tolerance through Association with Chi, an Accessory Clamp Loader factors to a blocked fork, without interfering with normal replication, is important for proper Protein. PLoS Genet 11(11): e1005651. doi:10.1371/ cell function, and it is a process that is not well understood. To study this process, we have journal.pgen.1005651 employed the chain-terminating nucleoside analog, 3’ azidothymidine (AZT) and the E. coli Editor: Lyle A. Simmons, University of Michigan, genetic system, for which replication and repair factors have been well-defined. By using UNITED STATES high-expression suppressor screens, we identified yoaA, encoding a putative helicase, and Received: April 28, 2015 holC, encoding the Chi component of the replication clamp loader, as genes that promoted Accepted: October 14, 2015 tolerance to AZT.
    [Show full text]
  • Tobias Viehboeck1, Harald Gruber-Vodicka2, Patrick Hyden3, Thomas Rattei3, Silvia Bulgheresi1
    Genomics of marine nematode symbionts Tobias Viehboeck1, Harald Gruber-Vodicka2, Patrick Hyden3, Thomas Rattei3, Silvia Bulgheresi1 - Laxus oneistus and Robbea hypermnestrae, two free-living nematodes (subfamily Stilbonematinae) live in a binary symbiotic association with chemoautotrophic, S-oxidizing Gammaproteobacteria, Candidatus Thiosymbion oneisti and Ca. T. hypermnestrae, respectively rchaea Biology and Ecogenomics - Each symbiont 9sattached to the nematode cuticle by one pole as to ensheath its host VIENNA © U. Dirks © N. Leisch © N. Leisch © N. Leisch Laxus oneistus 150 µm Robbea hypermnestrae 150 µm Ca. Thiosymbion oneisti 2 µm Ca. Thiosymbion hypermnestrae 1 µm Illumina - Nanopore Hybrid assembly Single-cell sequencing - A single-worm metagenome was sequenced on Illumina HiSeq 2x100 bp, - FACS & WGA-XTM at the Bigelow SCGC assembled (SPAdes 3.11), binned with gbtools - 10 single-amplified genomes for sequencing on Illumina NextSeq 550 - A symbiont isolate was sequenced on a MinION (Oxford Nanopore 2x150 bp (12M reads/cell) Technologies, ONT) - thereof 3 for ONT sequencing A A A A A A A A A A A B A A A A A A A A A A A B A A A A A A A A A A A B A A A A A A A A A A A B - The Illumina assembly was scaffolded with ultra long reads 03:23 04:02 08:55 02:03 05:30 03:22 03:41 02:00 06:56 02:08 02:48 02:04 02:05 02:04 03:30 02:04 02:29 B A A A A A A A A A A B A A A A A A A A A A A B B A A A A A A A A A A B A A A A A A A A A A A B 09:24 03:57 02:25 03:19 02:22 02:24 02:14 02:06 02:33 02:45 03:24 02:47 02:03 01:57 02:30 02:35 01:44 10:18
    [Show full text]
  • Structure, Function, and Therapeutic Targeting of SSB Protein Interactions
    Structure, function, and therapeutic targeting of SSB protein interactions By Aimee H. Marceau A dissertation submitted in partial fulfillment of the requirements of the degree of Doctor of Philosophy (Microbiology) at the UNIVERSITY OF WISCONSIN-MADISON 2012 Date of final oral examination: 09/21/2012 The dissertation is approved by the following members of the Final Oral Committee: James L. Keck, Professor, Biomolecular Chemistry Joseph Dillard, Professor, Medical Microbiology & Immunology Michael Cox, Professor, Biochemistry Michael G. Thomas, Professor, Bacteriology Warren E. Rose, Professor, Pharmacy i Structure, function, and therapeutic targeting of SSB protein interactions Aimee Helen Marceau Under the supervision of Professor James L. Keck University of Wisconsin-Madison ABSTRACT DNA unwinding creates single-stranded (ss) DNA intermediates that serve as templates for diverse cellular functions. Exposed ssDNA results in two specific problems for the cell; first, ssDNA is thermodynamically less stable than dsDNA, which leads to spontaneous formation of duplex secondary structures that impede genome maintenance processes. Second, relative to dsDNA, ssDNA is hypersensitive to chemical and nucleolytic attacks that can cause damage to the genome. These potential problems are solved by encoding specialized ssDNA-binding proteins (SSBs) that bind to and stabilize ssDNA structures required for essential genomic processes. The SSB protein in bacteria contains two functionally distinct regions; the N-terminal oligonucleotide/oligosaccharide binding (OB) fold is responsible for oligomerization and ssDNA ii binding, while the flexible amphipathic C-terminal tail (SSB-Ct) is the site of its essential interactions with many proteins involved in DNA processing and genome maintenance. SSB is directly involved with the replication process in bacteria.
    [Show full text]
  • Purification of Escherichia Coli Yoaa, a Putative Helicase Master's Thesis
    Purification of Escherichia coli YoaA, a Putative Helicase Master’s Thesis Presented to The Faculty of the Graduate School of Arts and Sciences Brandeis University Department of Biochemistry Dr. Susan Lovett, Advisor In Partial Fulfillment of the Requirements for the Degree Master of Science in Biochemistry by Mark Gregory May 2019 Copyright by Mark Gregory © 2019 Acknowledgements I would like to thank my advisor Dr. Susan Lovett. Thank you for sharing with me the incredible opportunity to work in her laboratory. I am grateful for your patience, optimism, encouragement and knowledge throughout the entirety of my work at your laboratory. Thank you to my mentor Vincent Sutera. Thank you for the time and support you gave me in allowing me to develop as a scientist. You instilled in me the importance of being organized in my thinking when carrying out my experiments and devising the next plans of action. These are qualities that go beyond the laboratory setting and will stay with me for the rest of my life. I would also like to thank everyone who worked with me in the Lovett lab. You were all very welcoming and provided a supportive environment during my experience. Thank you to all my friends. Special thanks to Elena for motivation and support. Thank you to the faculty in the Biochemistry Department for nourishing my love of biochemistry throughout my research. Thank you to my family for encouraging me and providing me with the support to always be ambitious. iii ABSTRACT Purification of Escherichia coli YoaA, a Putative Helicase A thesis presented to the Department of Biochemistry Graduate School of Arts and Sciences Brandeis University Waltham, Massachusetts By Mark Gregory All cells must maintain their genomic integrity to survive.
    [Show full text]
  • Architecture and Conservation of the Bacterial DNA Replication Machinery, an Underexploited Drug Target
    View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Research Online University of Wollongong Research Online Faculty of Science - Papers (Archive) Faculty of Science, Medicine and Health 2012 Architecture and conservation of the bacterial DNA replication machinery, an underexploited drug target Andrew Robinson University of Wollongong, [email protected] Rebecca J. Causer University of Wollongong, [email protected] Nicholas E. Dixon University of Wollongong, [email protected] Follow this and additional works at: https://ro.uow.edu.au/scipapers Part of the Life Sciences Commons, Physical Sciences and Mathematics Commons, and the Social and Behavioral Sciences Commons Recommended Citation Robinson, Andrew; Causer, Rebecca J.; and Dixon, Nicholas E.: Architecture and conservation of the bacterial DNA replication machinery, an underexploited drug target 2012, 352-372. https://ro.uow.edu.au/scipapers/2996 Research Online is the open access institutional repository for the University of Wollongong. For further information contact the UOW Library: [email protected] Architecture and conservation of the bacterial DNA replication machinery, an underexploited drug target Abstract "New antibiotics with novel modes of action are required to combat the growing threat posed by multi- drug resistant bacteria. Over the last decade, genome sequencing and other high-throughput techniques have provided tremendous insight into the molecular processes underlying cellular functions in a wide range of bacterial species. We can now use these data to assess the degree of conservation of certain aspects of bacterial physiology, to help choose the best cellular targets for development of new broad- spectrum antibacterials.
    [Show full text]
  • The Topoisomerase IV Parc Subunit Colocalizes with the Caulobacter Replisome and Is Required for Polar Localization of Replication Origins
    The topoisomerase IV ParC subunit colocalizes with the Caulobacter replisome and is required for polar localization of replication origins Sherry C. Wang*† and Lucy Shapiro*‡ *Department of Developmental Biology, Stanford University School of Medicine, Beckman Center B300, 279 Campus Drive, Stanford, CA 94305; and †Cancer Biology Program, Stanford Medical School, Stanford, CA 94305 Contributed by Lucy Shapiro, April 9, 2004 The process of bacterial DNA replication generates chromosomal motile swarmer cell and a stalked cell (Fig. 1A). In the swarmer topological constraints that are further confounded by simulta- cell, which is unable to initiate DNA replication, the origin of neous transcription. Topoisomerases play a key role in ensuring replication is located at the flagellated pole (1). DNA replication orderly replication and partition of DNA in the face of a continu- initiates when the swarmer cell differentiates into a stalked cell ously changing DNA tertiary structure. In addition to topological and replisome components assemble onto the replication origin constraints, the cellular position of the replication origin is strictly at the stalked cell pole (14). A copy of the replicated origin controlled during the cell cycle. In Caulobacter crescentus, the moves rapidly to the pole opposite the stalk in what is thought origin of DNA replication is located at the cell pole. Upon initiation to be an active process [see the companion article by Viollier et of DNA replication, one copy of the duplicated origin sequence al. (15) in this issue of PNAS], and as DNA replication proceeds, rapidly appears at the opposite cell pole. To determine whether the the replisome progresses from the stalked pole to the division maintenance of DNA topology contributes to the dynamic posi- plane (14).
    [Show full text]
  • Systematic Identification of Synthetic Lethal Mutations With
    Genes Genet. Syst. (2016) 91, p. 183–188 Systematic identification of synthetic lethal mutations with reduced-genome Escherichia coli: synthetic genetic interactions among yoaA, xthA and holC related to survival from MMS exposure Keisuke Watanabe, Kento Tominaga, Maiko Kitamura and Jun-ichi Kato* Department of Biological Sciences, Graduate Schools of Science and Engineering, Tokyo Metropolitan University, Minamiohsawa, Hachioji, Tokyo 192-0397, Japan (Received 22 October 2015, accepted 25 January 2016; J-STAGE Advance published date: 2 May 2016) Reduced-genome Escherichia coli strains lacking up to 38.9% of the parental chromosome have been constructed by combining large-scale chromosome deletion mutations. Functionally redundant genes involved in essential processes can be systematically identified using these reduced-genome strains. One large-scale chromosome deletion mutation could be introduced into the wild-type strain but not into the largest reduced-genome strain, suggesting a synthetic lethal interac- tion between genes removed by the deletion and those already absent in the reduced-genome strain. Thus, introduction of the deletion mutation into a series of reduced-genome mutants could allow the identification of other chromosome deletion mutations responsible for the synthetic lethal phenotype. We identified a synthetic lethality caused by disruption of nfo and xthA, two genes encoding apurinic/apyrimidinic (AP) endonucleases involved in the DNA base excision repair pathway, and two other large-scale chromosome deletions. We constructed temperature-sensitive mutants harboring quadruple-deletion mutations in the affected genes/chromosome regions. Using these mutants, we identified two multi-copy suppressors: holC, encoding the chi subunit of DNA polymerase III, and yoaA, encoding a putative DNA helicase.
    [Show full text]
  • Supplementary Materials: Modular Diversity of the BLUF Proteins and Their Potential for the Development of Diverse Optogenetic Tools
    Appl. Sci. 2019, 9, x FOR PEER REVIEW 1 of 10 Supplementary Materials: Modular Diversity of the BLUF Proteins and Their Potential for the Development of Diverse Optogenetic Tools Manish Singh Kaushik, Ramandeep Sharma, Sindhu KandothVeetil, Sandeep Kumar Srivastava and Suneel Kateriya Table S1. String analysis [1] output showing the details of query proteins, domains, interacting proteins and annotated functions. S. No. Query protein Domain Interacting Partner Annotation JD73_03740 C-di-GMP phosphodiesterase YeaP Diguanylate cyclase JD73_23680 Diguanylate cyclase JD73_23675 Diguanylate cyclase YdaM Diguanylate cyclase EAL (Diguanylate 1. JD73_24940 AriR Regulator of acid resistance cyclase) YcgZ Two-component-system connector protein HTH-type transcriptional regulator, MerR YcgE domain protein JD73_25605 Regulatory protein MerR GJ12_01945 Transcriptional regulator AMSG_00147 Phosphodiesterase AMSG_00905 Phosphodiesterase AMSG_01576 Uncharacterized protein Adenylyl cyclase-associated protein belongs to AMSG_01591 the CAP family AMSG_04591 DNA-directed RNA polymerase subunit beta CHD (class III AMSG_08774 Uncharacterized protein 2. AMSG_04679 nucleotydyl cGMP-dependent 3',5'-cGMP cyclase) AMSG_08967 phosphodiesterase A Adenylate/guanylate cyclase with GAF and AMSG_09378 PAS/PAC sensor 3,4-dihydroxy-2-butanone 4-phosphate AMSG_10048 synthase AMSG_11978 DNA helicase Hhal_0366 Multi-sensor hybrid histidine kinase Hhal_0474 CheA signal transduction histidine kinase Hhal_0522 Putative CheW protein Hhal_0934 CheA signal transduction histidine
    [Show full text]
  • Identification, Isolation, and Overexpression of the Gene Encoding the * Subunit of DNA Polymerase III Holoenzyme JEFFREY R
    JOURNAL OF BACTERIOLOGY, Sept. 1993, p. 5604-5610 Vol. 175, No. 17 0021-9193/93/175604-07$02.00/0 Copyright © 1993, American Society for Microbiology Identification, Isolation, and Overexpression of the Gene Encoding the * Subunit of DNA Polymerase III Holoenzyme JEFFREY R. CARTER,' MARY ANN FRANDEN,1 RUEDI AEBERSOLD,2 AND CHARLES S. McHENRY'* Department ofBiochemistry, Biophysics and Genetics, The University of Colorado Health Sciences Center, 4200 East Ninth Avenue, Denver, Colorado 80262,1 and The Biomedical Research Centre and Department ofBiochemistry, University ofBritish Columbia, Vancouver, British Columbia, Canada, V6T 1Z32 Received 26 April 1993/Accepted 16 June 1993 The gene encoding the 4 subunit of DNA polymerase m holoenzyme, holD, was identified and isolated by an approach in which peptide sequence data were used to obtain a DNA hybridization probe. The gene, which maps to 99.3 centisomes, was sequenced and found to be identical to a previously uncharacterized open reading frame that overlaps the 5' end of riml by 29 bases, contains 411 bp, and is predicted to encode a protein of 15,174 Da. When expressed in a plasmid that also expressed hoiC, holD directed expression of the * subunit to about 3% of total soluble protein. DNA polymerase III holoenzyme (referred to here as contribution of X and 4, to holoenzyme requires purification holoenzyme) is the 10-subunit replicative enzyme of Esche- of large quantities of each subunit. In this report, we present richia coli. Several biochemical properties distinguish this a vital step toward this objective: the identification, isola- polymerase from the nonreplicative polymerases of E.
    [Show full text]
  • Exploring Tripartite Interaction Between Yoaa, Holc, and SSB, Required for Repair of DNA Damage Made by AZT
    Exploring tripartite interaction between YoaA, HolC, and SSB, required for repair of DNA damage made by AZT Una Milovanovic, Sneha Sathish, Margaret Hibnick, Mentor: Doctor Linda Bloom Department of Biochemistry and Molecular Biology, College of Medicine, University of Florida, Gainesville, FL Introduction The structure of DNA is constantly being challenged by chemicals that are naturally present in cells including oxygen and water. It has been estimated that 20,000 DNA damaging events occur within a cell in a 24 hour period. If those damages do not get fixed, the genetic code will be altered and chromosomes may break. This issue is addressed by cell mechanisms where the damaged bit of DNA is removed and the undamaged DNA strand is used as a template to remake the damaged strand. However, there is some damage that does not get repaired prior to DNA replication. In this case, cell have to conduct special mechanisms to either fix the damaged DNA during replication or bypass the damage and fix it later. Our collaborators in the Lovett laboratory at Brandeis University use genetic approaches in Escherichia coli, a model organism, to discover processes that can repair DNA damage during replication. They recently discovered a pathway that requires at least two proteins, HolC and YoaA, to give cells tolerance to DNA damage created by 3’-azido- 3’-thymidine (AZT). AZT is a chain terminator so that when it is incorporated into DNA by a DNA polymerase, the DNA polymer cannot be extended any further. Thus, DNA replication is blocked. When the polymerase stops, single-stranded DNA starts to accumulate and is bound by a protein called single-stranded DNA binding protein or SSB.
    [Show full text]